Designing laser-modified surface structures on titanium alloy custom medical implants using a hybrid manufacturing technology.

Osteosarcoma (OS) is a primary bone sarcoma with a high recurrence rate and poorer prognosis. The application of natural agents in combinational therapies can increase the efficacy of treatment and decrease the side effects. Herein, we aimed to evaluate the effects of Thymoquinone (TQ) combined with Cisplatin on apoptosis and its underlying mechanisms in the Saos-2 cells. The effects of TQ and Cisplatin on Saos-2 cell viability were measured using an MTT assay. Western blotting was applied for the measurement of γH2AX protein expression. The expression levels of 8-Hydroxy-2'-deoxyguanosine (8-oxo-dG) were evaluated by enzyme-linked immunosorbent assay (ELISA). DCFH-DA fluorescence dye was used to detect reactive oxygen species (ROS) formation. For evaluation of apoptosis, flow cytometry was employed. TQ dramatically promotes the cytotoxic effects of Cisplatin. TQ considerably enhanced the expression levels of 8-oxo-dG and γ-H2AX in Saos-2 cells. After TQ treatment, ROS levels were increased; furthermore, TQ treatment resulted in the potentiation of Cisplatin-induced apoptosis in Saos-2 cells compared to either TQ or Cisplatin treated cells. In general, TQ plus Cisplatin resulted in potentiated cellular cytotoxicity by increasing ROS level and inducing oxidative DNA damage, leading to the potent induction of apoptosis in tumor cells.

Effects of five different resin-based sealers on L929 and Saos-2 cell viability

Circular RNAs (circRNAs) are emerging as important regulators in human disease, but their function in osteoporosis (OP) is not sufficiently known. The aim of this study was to identify the possible molecular mechanism of circ_KIAA0922 in osteogenic differentiation of Saos-2 cells in vitro and the interactions among circ_KIAA0922, miR-148a-3p, and SMAD family member 5 (SMAD5). Circ_ KIAA0922, miR-148a-3p, and SMAD5 were overexpressed by transient transfection. Dual-luciferase reporter assay system was used to analyze the combination among circ_KIAA0922, miR-148a-3p, and SMAD5. In addition, the levels of circ_KIAA0922, miR-148a-3p, SMAD5, osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) were detected using RT-qPCR or western blot analysis. Alizarin red staining was performed to analyze the degree of osteogenic differentiation under the control of circ_KIAA0922, miR-148a-3p, and SMAD5. We found that circ_KIAA0922 knockdown inhibited the proliferation and osteogenic differentiation of Saos-2 cells. Circ_KIAA0922 directly targeted miR-148a-3p, and miR-148a-3p inhibition reversed the effects of circ_KIAA0922 knockdown on the proliferation and osteogenic differentiation of Saos-2 cells. Overexpression of SMAD5 promoted the proliferation and osteogenic differentiation of Saos-2 cells and attenuated the inhibitory effect of miR-148a-3p on cell proliferation and osteogenic differentiation. In conclusion, circ_ KIAA0922 facilitated Saos-2 cell proliferation and osteogenic differentiation via the circ_KIAA0922/ miR-148a-3p/ SMAD5 axes in vitro, thus providing insights into the mechanism of osteogenic differentiation by circ_ KIAA0922.

The chemical composition of biomaterials can drive their biological responses; therefore, this in vitro study aimed to evaluate the proteomic profile of the salivary pellicle formed on titanium (Ti) alloys containing niobium (Nb) and zirconium (Zr). The experimental groups consisted of Ti35NbxZr (x = 5 and 10 wt%) alloys, and commercially pure titanium (cpTi); titanium aluminium vanadium (Ti6Al4V) alloys were used as controls. The physical and chemical characteristics of the Ti materials were analysed. The proteomic profile was evaluated by liquid chromatography coupled with tandem mass spectrometry. Bacterial adhesion (2 h) of mixed species (Streptococcus sanguinis and Actinomyces naeslundii) was investigated as colony-forming units (n = 6). This paper reports the finding that salivary pellicle composition can be modulated by the composition of the Ti material. The Ti35NbxZr group showed a significant ability to adsorb proteins from saliva, which can favour interactions with cells and compatibility with the body.

Simple SummaryCalcium silicate-based cements are successfully applied in the different fields of endodontics and vital pulp therapy. To better assess the properties of these bioactive materials, the present in vitro and in vivo study aimed to compare the biocompatibility and antibiofilm properties of ProRoot MTA and Biodentine. Human osteogenic sarcoma (Saos-2) cells were cultured in the presence of both materials and evaluated. Moreover, the bioactive cements were in vivo applied to perform vital pulp therapy on immature permanent teeth affected by reversible pulpitis. Saos-2 cells’ viability was slightly greater in the presence of ProRootMTA than Biodentine and cells would grow in a better way on ProRootMTA disks than on Biodentine ones. Moreover, ProRootMTA showed a powerful antibiofilm effect towards Streptococcus mutans. The in vitro results were clinically supported by a 100% success rate after 2 years of follow-up.Calcium silicate-based cements have reached excellent levels of performance in endodontics, providing predictable and successful results. To better assess the properties of these bioactive materials, the present study aimed to compare the biocompatibility and antibiofilm properties of ProRoot MTA and Biodentine. Human osteogenic sarcoma (Saos-2) cells were cultured on ProRoot MTA and Biodentine samples or in the presence of both cement extracts. Cell viability assay, measurement of reactive oxygen species (ROS), immunofluorescence analysis, as well as morphological evaluations were conducted. Moreover, Streptococcus mutans was used to assess the biofilm forming ability on ProRoot MTA and Biodentine disks. Finally, both cements were applied in vivo to treat immature permanent teeth affected by reversible pulpitis. Results: Cell viability assay demonstrated that Saos-2 cells had a dose- and time-dependent cytotoxicity to both analyzed cements, although cells exposed to ProRoot MTA showed a better cell vitality than those exposed to Biodentine (p < 0.001). Both cements demonstrated ROS production while this was greater in the case of Biodentine than ProRoot MTA (p < 0.001). Immunofluorescence images of the cytoskeleton and focal adhesions showed no differences in Saos-2 cells grown in the presence of ProRoot MTA eluate; whereas in the Biodentine groups, cells showed a morphology and focal adhesions more similar to that of the control sample, as the eluate concentration decreased. Morphological analysis revealed that Saos-2 cells were more flattened and exhibited better spreading when attached to ProRoot MTA disks than to Biodentine ones. The antibiofilm properties showed a time-dependent powerful inhibition of S. mutans superficial colonization and an antibiofilm effect of both cements. Clinically, complete root formation of the treated elements was achieved using the two studied cements, showing stable results over time. ProRoot MTA and Biodentine was demonstrated to be biocompatible and to possess antibiofilm properties. Their clinical application in vital pulp therapy provided successful outcomes after 2 years of follow-up.

Resveratrol has been shown to have antineoplastic effects in vivo and in vitro. However, the effect of resveratrol on the hypoxia-enhanced proliferation and invasion of osteosarcoma cells remains unclear. In this study, we investigated the role of resveratrol on regulating proliferation and invasion of osteosarcoma cells under hypoxic conditions. Saos-2 cells were cultured under controlled hypoxic conditions (3% O2) or normoxic conditions. Resveratrol (50 µM) was added in the medium, and hypoxia inducible factor-1α (HIF-1α) siRNA was used to inhibit HIF-1α transcription. Proliferation of Saos-2 cells was evaluated by the methabenzthiazuron (MTT) assay. The invasive ability of Saos-2 cells was determined by a Transwell assay. HIF-1α, E-cadherin and vimentin protein levels were detected by western blot analysis. HIF-1α, E-cadherin and vimentin mRNA levels were assessed by RT-PCR. Compared to the control group, hypoxia significantly increased the proliferation rate and invasive ability of Saos-2 cells. Moreover, hypoxia markedly increased the E-cadherin level and decreased vimentin expression. However, resveratrol or HIF-1α silencing reverted all the above effects of hypoxia in Saos-2 cells. Moreover, resveratrol inhibited HIF-1α protein accumulation without affecting the HIF-1α mRNA level. These data suggest that resveratrol can inhibit the hypoxia‑enhanced proliferation, invasion and epithelial to mesenchymal transition process in osteosarcoma via downregulation of the HIF-1α protein. Thus, HIF-1α may be a promising drug target of resveratrol in the context of development of anticancer therapy for human osteosarcoma.

The term ‘vaccination’ describes the induction of protective immune responses against infectious diseases, but is also used to define antigen-specific forms of immunotherapy for allergy, cancer and autoimmunity. Successful vaccination requires either immune modulation or the induction of robust specific immunity to several disease-causing antigens. However, natural antigen sources may contain greatly varying amounts of these antigens and some of them may exhibit low immunogenicity. An approach for overcoming the latter problems has been developed for allergy vaccines recently. This approach is based on the genetic engineering of hybrid molecules, consisting of several major disease-eliciting antigens/epitopes. Such hybrid molecules can be built to include the most relevant epitopes of complex antigen sources. Moreover, fusion of different antigens in the form of hybrid molecules strongly increases their immunogenicity. The hybrid approach can also be used for the generation of mosaic antigens with altered immunological properties, which consist of re-shuffled antigen pieces. We exemplify the use of hybrid technology for the generation of new allergy vaccines and discuss its potential applicability for the development of vaccines for infectious diseases, cancer and autoimmunity.

Exploring the molecular mechanism of linagliptin in osteosarcoma cell lines for anti-cancer activity

The standard flow cytometry method for viability testing using 7-aminoactinomycin D (7-AAD) determines cells in necrosis and late apoptosis. The colony-forming unit (CFU) assay, which evaluates the proliferation ability of HSCs, is also used in graft quality assessment despite known deficiencies that make this assay impractical in routine clinical settings. The aim was to compare the effectiveness of the flow cytometry 7-AAD/annexin V method with the 7-AAD method in assessing the quality of HSCs in autologous and allogeneic peripheral blood stem cell (PBSC) products. Thirty autologous and 30 allogeneic fresh and thawed cryopreserved PBSC products were included in this study. The viability of HSCs was determined using the 7-AAD method and 7-AAD/annexin V method on a flow cytometer, while their clonogenic capacity was assessed by CFU assay. There was an excellent correlation for CD34+ cell viability between the 7-AAD and the 7-AAD/annexin V method for fresh samples (Rs = 0.930, p < 0.001) and a good correlation for thawed PBSC samples (Rs = 0.739, p < 0.001). Excellent correlation was observed for post-thaw CD34+ cell recovery between the two methods for viability (Rs = 0.980, p < 0.001). Statistical analysis showed a weak correlation between CFU-GM recovery and CD34+ cell recovery, regardless of which viability testing method was used (7-AAD method p = 0.021, Rs = 0.298; 7-AAD/annexin V method p = 0.029, Rs = 0.282). Results of this study showed that in the quality assessment of cryopreserved PBSC product viability, the 7-AAD/annexin V method had no added value compared to the 7-AAD method, which was suitable enough for routine quality control of cryopreserved autologous and allogeneic PBSC samples.

A porous Ti6Al4V implant that is manufactured using selective laser melting (SLM) has broad potential applications in the field of orthopaedic implants. The pore structure of the SLM porous Ti6Al4V implant allows for cell migration and osteogenic differentiation, which is favorable for bone ingrowth and osseointegration. However, it is unclear whether the pore structure and partially melted Ti6Al4V particles on a SLM porous Ti6Al4V implant will increase bacterial adhesion and, perhaps, the risk of implant-related infection. (1) Is there more bacterial adhesion and colonization on SLM porous Ti6Al4V implants than on polished orthopaedic implants? (2) Do partially melted Ti6Al4V particles on SLM porous Ti6Al4V implants reduce human bone mesenchymal stem cells (hBMSCs) adhesion, viability, and activity? To determine bacterial adhesion and biofilm formation, we incubated five different Ti6Al4V discs (polished, grit-blasted, plasma-sprayed, particle SLM porous, and nonparticle SLM porous discs) with methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli. Bacterial coverage on the surface of the five different Ti6Al4V discs were evaluated based on scanning electron microscopy (SEM) images quantitatively. In addition, a spread-plate method was used to quantitatively evaluate the bacterial adhesion on those implants. The biofilm formation was stained with crystal violet and semi-quantitatively determined with a microplate reader. The morphology and adhesion of hBMSCs on the five Ti6Al4V discs were observed with SEM. The cell viability was quantitatively evaluated with a Cell Counting Kit-8 assay. In addition, the osteogenic activity was determined in vitro with a quantitatively alkaline phosphatase activity assay and alizarin-red staining. For semiquantitative analysis, the alizarin-red stained mineralized nodules were dissolved and determined with a microplate reader. The polished discs had the lowest MRSA adhesion (8.3% ± 2.6%) compared with grit-blasted (19.1% ± 3.9%; p = 0.006), plasma-sprayed (38.5% ± 5.3%; p < 0.001), particle (23.1% ± 2.8%; p < 0.001), and nonparticle discs (15.7% ± 2.5%; p = 0.003). Additionally, when comparing the two SLM discs, we found that particle discs had higher bacterial coverage than nonparticle discs (23.1% ± 2.8% versus 15.7% ± 2.5%; p = 0.020). An E. coli analysis showed similar results, with the higher adhesion to particle SLM discs than to nonparticle discs (20.7% ± 4.2% versus 14.4% ± 3.6%; p = 0.011). In addition, on particle SLM porous discs, bacterial colonies were localized around the partially melted Ti6Al4V particles, based on SEM images. After a 7-day incubation period, the cell viability in the particle group (optical density value 0.72 ± 0.05) was lower than that in the nonparticle groups (optical density value: 0.87 ± 0.08; p = 0.003). Alkaline phosphatase activity, as a marker of osteogenic differentiation, was lower in the particle group than in the nonparticle group (1.32 ± 0.12 U/mL versus 1.58 ± 0.09 U/mL; p = 0.012). Higher bacterial adhesion was observed on SLM porous discs than on polished discs. The partially melted Ti6Al4V particles on SLM porous discs not only enhanced bacterial adhesion but also inhibited the osteogenic activity of hBMSCs. Postprocessing treatment is necessary to remove partially melted Ti6Al4V particles on an SLM implant before further use. Additional studies are needed to determine whether an SLM porous Ti6Al4V implant increases the risk of implant-related infection in vivo. As implants with porous Ti6Al4V made using SLM are being designed, our preliminary findings suggest that postprocessing treatment is needed to remove partially melted Ti6Al4V particles before further use. In addition, the depth of the porous structure of the SLM implant should not exceed the maximum depth of bone ingrowth because the host immune defense cannot prevent bacterial adhesion without integration.

The desire for larger and larger format arrays for astronomical observatories -- both ground and space based -- has fueled the development of detector, readout, and hybrid Focal Plane Array (FPA) technology that has paved the way for later development of tactical and strategic arrays for military applications. Since 1994, Raytheon has produced megapixel readouts and FPAs for Infrared Astronomy. In 1999 Raytheon demonstrated a revolutionary approach to photolithography called Reticle Image Composition Lithography (RICL) that opened the door to very large format FPAs in state of the art sub-micron CMOS processes. The first readout processed using the patented RICL technique was a 4.2 megapixel readout for astronomy. We present the design and performance of several 4.2 megapixel (2048 x 2048) readout arrays for visible and infrared astronomy applications. The first of these arrays are fabricated in a workhorse 2 μm CMOS process that is optimized for low temperature operation (down to as low as 6 Kelvin). Most recently Raytheon has developed a scaleable 2,048 x 2,048 high density array for several ground based astronomical applications. This array can be manufactured in any m x n multiple of a basic 1024 (V) x 512 (H) pixel array core. The primary design is a 2 x 4 array to yield a 2,048 x 2,048 format array. This same design can be extended to at least a 4,096 x 4,096 format array -- an incredible 16.7 megapixel array! These readouts are compatible with a wide range of detector types including InSb, HgCdTe, and Si detectors. The use of hybrid technology -- even for the visible wavebands -- allows 100% optical fill factors to be achieved. The design and performance of these megapixel class detectors, readouts, and FPAs will be presented.

A novel device, piezoelectric 11 Gracey curet tip, reportedly combines benefits of a piezoelectric device and manual curet. The primary objective of this study was to compare root surface roughness outcomes between traditional manual curets and piezoelectric devices, as compared with this novel device. The secondary aim was to assess the level of adhesion of Streptococcus mutans on the root surface after instrumentation. The groups consisted of the following: 1) Gracey curet; 2) piezoelectric scaler; 3) piezoelectric 11 Gracey curet tip; and 4) untreated control. Root specimens were obtained from extracted human teeth and randomly assigned to each group. Surface roughness measurements (Ra and Rz) were taken with a profilometer before and after instrumentation. After instrumentation, root specimens were inoculated with S. mutans and biofilm was dislodged. Various dilutions of resuspended biofilm were incubated on blood agar plates and colony forming units (CFU) values were measured. The experimental device resulted in significantly lower Ra and Rz compared with other groups (P<0.01), and the Gracey curet was significantly lower than the piezoelectric tip and untreated control (P<0.05). The Gracey curet and experimental device tip had significantly lower CFU values compared with the control (P<0.05). There were no significant CFU value differences between the Gracey curet and both the piezoelectric and experimental device tips. There were no significant CFU differences between piezoelectric tip and both experimental device and control. There was no correlation between Ra and CFU values for Gracey curet, piezoelectric tip, or the control. However, correlation between Ra values and CFU approached significance for the experimental device (correlation=0.66, P=0.05). Piezoelectric 11 Gracey curet tip is effective at resulting in a significantly smoother surface compared with traditional piezoelectric and hand instruments. CFU values with piezoelectric 11 Gracey curet tip were significantly lower than non-instrumented surfaces, but there were no significant differences compared with conventional methods.

It has been speculated that periodontal probes can transmit periodontal pathogens from site to site. The purpose of this study was to evaluate the potential for bacterial transmission as a function of periodontal probe design. Four different periodontal probes were used to measure probing depths ranging from 0 to 3 mm and > or = 4 mm. Following measurement of each pocket, probes were transported to a laboratory and cultured on blood agar, and colony forming units (CFUs) were determined for total aerobic, anaerobic, and dark-pigmented colonies (DPCs). Eight randomly selected probe tips, representing each probe type and probing depth category, were selected for examination by scanning electron microscopy (SEM) to determine distribution and morphotypes of adhering microbes. Results showed no statistically significant main effect for probes with respect to CFUs. However, there was a statistically significant increase in CFUs for deeper pockets when compared to pockets 0 to 3 mm. SEM observations were consistent among groups, regardless of probe design or probing depth. All probes exhibited a rough surface texture. Microbes were observed as single organisms or in aggregates and were adherent along the entire length of the probe up to the maximum probing depth. There was no difference in CFUs obtained as a function of periodontal probes. The rough surfaces of the probes used in this study likely promoted bacterial adherence through two different mechanisms: through scraping of the pocket walls by the roughened surfaces of the probe and by the high surface-free energy of the metal probes, facilitating bacterial adherence.

IntroductionAs per FACT standards, Cord Blood Banks (CBB) have to verify that they provide a safe and effective product for transplant. Validated processing methods and storage conditions are the first steps towards this goal, but what are the final steps to monitor the quality of the cord blood units (CBU) released for transplant?ObjectivesWe present here a retrospective analysis of our Program’s final verification process to meet this FACT requirement.MethodsCBU shipped between January 2018 and December 2022 were reviewed for out of specification events. The following Quality Control (QC) parameters were measured on attached segments before shipment: total nucleated cells (TNC), viable CD34 cells (vCD34), and colony forming units (CFU). Confirmed date of infusion and engraftment data were obtained from CIBMTR.ResultsA total of 619 CBU were shipped during the study period. 550 had a segment analyzed at the time of shipment. Average post-thaw CD34 viability on the segments was 93.5% (Table 1). Average recoveries for TNC, viable CD34 and CFU were 83.3%, 64.90% and 60.86%, respectively (Table 1). 6 (1%) CBUs were found to have CD34 viability lower than FACT requirement (≥70%) (Table 1). Investigations were performed for each CBU, concluding that these were technical issues with no repercussion on the final product. No segments demonstrated absence of CFU growth. 544 CBUs had an infusion date recorded by CIBMTR (Table 1). 250 of the CBU infused had engraftment data, and 242 engrafted. 8 (3%) CBU transplants resulted in non-engraftment. All had QC results within expected range (Table 1).Table 1.Quality control testing and outcome dataQuality control testingValues and Results Post-thaw segment CD34 viability (average, range, n)95.27% (37.75 – 100), n=777a Post-thaw segment TNC recovery (average, range, n)83.35% (42.6 – 128.6), n=550 Post-thaw segment viable CD34 recovery (average, range, n)64.90% (14.5 – 134.7), n=550 Post-thaw segment CFU recovery (average, range, n)60.94% (10.6 – 155.8), n=516b Outside of acceptance criteria CD34 viability <70%n=6c 3 CBU, each cryopreserved in 2 bags, second bag within expected rangeAll infused2 patients engrafted1 patient had no outcome data 3 CBU, each cryopreserved in one bag1 infused, no outcome data1 infusion pending1 non infused (back up CBU infused) No CFU growthn=0Outcome data,d units infused (n=544) Adults321 (59%) Children223 (41%) Diagnosis Malignant disease472 (87%) Nonmalignant disease72 (13%) Single331 (61%) Multiple Double CBU 184 (34%) CBU + PBSC26 (4%) >3 CBU3 (1%) Match 3/62 (0.4%) 4/6316 (58.1%) 5/6184 (33.8%) 6/642 (7.7%) CBU storage time, average (range)7.5 years (7 months – 19.5 years) Cell doses TNC x10e7/kg (median, range)3.4 (0.7 – 56.7) CD34 x10e3/kg (median, range)205 (16 – 4856) Engraftment analysis (n=250) Engraftment242 (96.8%) Non engraftment8 (3.2%)a777 segments were assessed for CD34 viability (227 CBU were preserved into 2 bags, 323 CBU into one bag)b516 CBU were assessed for CFU recovery (34 CBU had not post-processing CFU testing)cLow viability events trigger an extensive review of manufacturing and distribution data of the CBU, as well as repeat testing of the flow cytometry. For all 6 CBU, repeat testing resulted in similar values; no issues were identified during data review and investigation pointed towards a technical problem at the time of thaw, with no root cause identified.d7 CBU had QC at shipment within acceptable range. 1 CBU had QC 4 years prior (at HLA confirmation) within acceptable range.DiscussionOur QC results confirm the continued stability of the CBU until shipment. The small subset of CBU with out of specification results were linked to technical issues and did not affect the outcome of transplant. Outcome data is another way of assessing the quality of the CBUs, even though it may be influenced by many factors out of the CBB’s responsibility (such as diagnosis, conditioning, multiple transplants, etc…). The combined analysis of the two datasets is a good way for a CBB to ensure processes are performing as expected over time and monitor the quality of the final product.

Selective estrogen receptor modulators (SERMs) are currently in use in the hormonal therapy of breast cancer. In that respect, a new hormone-related approach is the therapeutical inhibition of steroid sulfatase (STS), which converts inactive, sulfated steroids into active hormones. We investigated the potential of 6-EO-14, a non-steroidal STS inhibitor with SERM potential. The latter compound, which exhibits a sulfamate moiety, releases the phenol derivative 8-EO-14 after the irreversible inhibition of STS. STS was inhibited by 6-EO-14 (IC50 = 0.3 μM), but not 8-EO-14, in HEK-293 cells transfected with an STS expression vector. The SERM potential of 8-EO-14 was assessed in osteoblast-like Saos-2 cells by investigating its effect on cell proliferation and on the activity of alkaline phosphatase (ALP), a specific differentiation marker. Saos-2 cell proliferation was increased by 21 % following 8-EO-14 addition (1 μM), and 8-EO-14 induced ALP activity (31 % increase at 0.1 nM) via estrogen receptor alpha (ERα) similarly to the SERM raloxifene. As compared to estradiol (E2) (100 %), the relative binding affinity of 6-EO-14 and 8-EO-14) for ERα was found to be weak (0.09 and 0.01 %, respectively). When assessed in two estrogen-dependent human breast cancer cell lines (MCF-7 and T-47D), 8-EO-14 did not support MCF-7 cell proliferation, whereas both 8-EO-14 and 6-EO-14 exhibited estrogen-like growth stimulation in T-47D cells. These two compounds were also unable to block E2-induced cell proliferation, suggesting their lack of antiestrogenic activity. Despite the known potency of 6-EO-14 as an STS inhibitor, the observed trophic activity of this new scaffold towards ERα-positive cells needs to be carefully considered prior to its potential utilization as a therapeutic agent.